Our long term goal in this project is to understand the mechanisms that regulate pre-mRNA alternative splicing. The majority of human genes express multiple mRNAs resulting in the expression of functionally diverse isoforms or the on-off regulation of gene expression. It is now clear that alternative splicing is often highly regulated and constitutes an important component of the integrated networks that control gene expression. Splicing is regulated by RNA binding proteins that bind specific sequence motifs within the pre-mRNA, usually local to the regulated splice site(s). Of particular interest in this proposal are auxiliary splicing regulators that are not involved in recognition of constitutive exons and appear to only transiently associate with the basal splicing machinery (the spliceosome) to regulate alternative splicing. The focus of this proposal is the molecular mechanisms by which bound splicing regulators communicate with the spliceosome to activate splicing. For the few splicing regulators for which details are known, evidence indicates that different regulators utilize diverse mechanisms of regulation. In fact, differences exist in the mechanisms by which one regulator controls splicing of different pre-mRNA targets. We will continue our analysis of CUGBP and ETR-3 like factors (CELF) and muscleblind-like (MBNL) protein families as direct regulators of alternative splicing. These different protein families often regulate the same pre-mRNA targets, do so antagonistically by binding to distinct sequence motifs, are activators and repressors of different splicing events, and are key regulators of postnatal splicing transitions that occur during normal heart and skeletal muscle development. Disruption of their splicing activities significantly contributes to the pathogenesis of myotonic dystrophy, the second most common form of muscular dystrophy, making them directly relevant to human disease. We will continue our analysis of the molecular interactions that these proteins form to communicate to the spliceosome. A major component of this proposal is comparison of mechanisms utilized by CELF and MBNL proteins. From this broad-based analysis we expect to obtain a breadth of understanding that will be widely applicable to an understanding of splicing regulation. The knowledge gained will be directly relevant to understanding mechanisms of disease as well as providing therapeutic approaches to reverse or circumvent disease processes. PUBLIC HEALTH RELEVANCE: Alternative splicing is a primary mechanism regulating gene expression the disruption of which results in disease. The two protein families that are the subject of this investigation are involved in the pathogenic mechanism of myotonic dystrophy, the most common form of adult onset muscular dystrophy. Understanding the normal mechanisms of regulation is crucial for developing strategic therapeutic approaches.